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R K Carlin

Publications and source records attributed to R K Carlin.

At least 19 recordsLinked to original sources
PubMed1986

Characteristics of a Ca2+/calmodulin-dependent binding of the Ca2+ channel antagonist, nitrendipine, to a postsynaptic density fraction isolated from canine cerebral cortex.

Synaptic membrane (SM) and postsynaptic density (PSD) fractions isolated from the cerebral cortex (CTX) and cerebellum (CL) of the canine brain were found to contain one class of specific nitrendipine binding sites. The specific binding constants were: CTX-SM, Kd = 110 pM (Bmax = 126 fmol/mg protein); CTX-PSD, Kd = 207 pM (Bmax = 196 fmol/mg); CL-SM, Kd = 100 pM (Bmax = 65 fmol/mg); CL-PSD, Kd = 189 pM (Bmax = 80 fmol/mg). Treatment of the CTX-SM and CTX-PSD fractions with 0.5% deoxycholate and 1.0% N-lauroyl sarcosinate removed 88-91% and 42-51% of the nitrendipine binding, respectively, indicating that the major nitrendipine binding present in the SM fractions are of non-synaptic origin. Moreover, the percentages of total protein and specific nitrendipine binding removed from PSDs by these detergents were similar, indicating no preferential dissociation of the latter, and suggesting that the receptor protein is firmly bound and is probably an intrinsic component of the PSD fraction. Both Ca2+ and calmodulin were found to be important for the binding of nitrendipine to the CTX-SM and CTX-PSD fractions since: R24571, a calmodulin antagonist, was found to inhibit nitrendipine binding to the CTX-SM and CTX-PSD fractions with IC50 values of 1.1 microM and 0.9 microM, respectively; removal of Ca2+ from the CTX-SM and CTX-PSD fractions with 0.2 mM EGTA resulted in losses of specific nitrendipine binding of 80 and 90%, respectively; Ca2+ alone restored nitrendipine binding to EGTA-pretreated CTX-SM fractions and not to CTX-PSD fractions, with the latter needing both Ca2+ and calmodulin to restore nitrendipine binding; EGTA treatment removed 14-16% and 89-91% of nitrendipine bound to the CTX-SM and CTX-PSD fractions, respectively, suggesting that calmodulin (but not Ca2+) is needed to maintain the nitrendipine-nitrendipine receptor-calmodulin complex; Ca2+-reconstituted EGTA-pretreated CTX-SM fractions and the Ca2+ plus calmodulin-reconstituted EGTA-pretreated CTX-SM and CTX-PSD fractions were found to have similar binding constants to those for the corresponding native, untreated fractions; and the Ca2+/calmodulin dependency on nitrendipine binding was similar to the well-known Ca2+/calmodulin dependency on phosphorylation in EGTA-pretreated PSD fractions. It needed much less Ca2+ to saturate Ca2+/calmodulin-dependent phosphorylation of the pretreated CTX-PSD fractions than the nitrendipine binding. Yet, less calmodulin was needed to saturate nitrendipine binding than the phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Comparison of proteins involved with cyclic AMP metabolism between synaptic membrane and postsynaptic density preparations isolated from canine cerebral cortex and cerebellum.

Synaptic membrane and postsynaptic density (PSD) fractions isolated from canine cerebral cortex and cerebellum were assayed for the following proteins: adenylate cyclase and phosphodiesterase (PDE) activities against cyclic AMP and cyclic GMP, the regulatory subunit of the cyclic AMP-dependent protein kinase, and the substrate proteins for this kinase. The results were expressed on the basis of both the protein content of the fractions and the number of synapses in the synaptic membrane fractions. The number of synapses on a constant protein content basis was about three times higher in the cerebral cortex synaptic membrane fraction than in the comparable cerebellar fraction. Adenylate cyclase activity was from 3.4 to 5.6 times higher in the cerebral cortex membrane fraction than in the cerebellar membrane fraction based on protein content but only slightly higher based on synapse counts. PSD fractions had no adenylate cyclase activity. The cyclic AMP-PDE activity was from 17 to 27 times higher in the cerebral cortex membrane fraction than in the cerebellar membrane fraction based on protein content, and about five times higher based on synapse counts. By doing PDE histochemistry at the electron microscopy level it was found that all the cerebral cortex PSDs in the isolated fraction contained PDE activity, none being found associated with the broken-up material in the fraction. The amount of the regulatory subunit of the cyclic AMP-dependent protein kinase was about equal in the two fractions based on protein, but about one-third lower in cerebral cortex fraction than in cerebellar fractions. In the cerebral cortex membrane fraction the primary substrate for the cyclic AMP-dependent protein kinase is synapsin I, with much lower amounts in the cerebellar membrane fraction. The PSD fraction from the two sources also showed these differences in synapsin I content. In the cerebellar membrane fraction, the primary substrate for the enzyme is a approximately 245,000 Mr protein not found in the cerebral cortex membrane fraction. The findings that the turnover of cyclic AMP is much higher in cerebral cortex synapses than in cerebellar synapses, and that differences are found between the cerebral cortex and cerebellum with regard to the substrate proteins for the cyclic AMP-dependent protein kinase indicate a divergence in the effect of cyclic AMP between cerebral cortex and cerebellar synapses.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

Similarities between the Mr 245,000 calmodulin-binding protein of the dogfish erythrocyte cytoskeleton and alpha-fodrin.

The Mr 245,000 calmodulin-binding protein of the dogfish erythrocyte cytoskeleton (D245) has been compared with human erythrocyte spectrin and mammalian brain fodrin [J. Levine and M. Willard (1981) J. Cell Biol. 90, 631-643]. Mammalian erythrocyte alpha-spectrin, brain alpha-fodrin, and D245 are all localized in the cell surface-associated cytoskeleton, and have similar molecular weights. Like mammalian erythrocyte spectrin, D245 was extracted from erythrocyte ghosts under low-ionic-strength conditions. However, D245 failed to bind an antibody which reacted strongly with both subunits of human erythrocyte spectrin. Unlike mammalian erythrocyte alpha- and beta-spectrin, D245 bound calmodulin in the absence of urea both in a "gel-binding" assay and in situ using azidocalmodulin [D.C. Bartelt, R.K. Carlin, G.A. Scheele, and W.D. Cohen (1982) J. Cell Biol. 95, 278-284]. Striking similarities were noted between D245 and alpha-fodrin in that both exhibited (a) comparable calcium-dependent calmodulin binding properties, (b) strong reactivity with two different anti-fodrin antibody preparations, (c) similar reactivity with antibody to brain CBP-I, now believed to be fodrin, (d) proteolytic degradation yielding an Mr 150,000 calmodulin-binding fragment, and (e) lack of reactivity with an anti-spectrin antibody. A protein with calmodulin-binding and anti-fodrin-binding properties similar to D245 was detected in cytoskeletal preparations of chicken erythrocytes. Moderate and consistent cross-reactivity of anti-fodrin with human erythrocyte alpha-spectrin was also observed. The data indicate that D245 is functionally and immunologically more closely related to alpha-fodrin than to alpha-spectrin of the mammalian erythrocyte.

Animals↗
PubMed1984

Characterization of Na+-independent GABA and flunitrazepam binding sites in preparations of synaptic membranes and postsynaptic densities isolated from canine cerebral cortex and cerebellum.

The binding of [3H]GABA and [3H]flunitrazepam was performed with synaptic membranes and post-synaptic densities (PSDs) isolated from canine cerebral cortex and cerebellum. Two GABA binding sites were found with cerebral cortex membranes but only one with cerebellar membranes. PSDs isolated from these showed only single binding sites, with cerebellar PSDs exhibiting lower KD values and a larger concentration of sites than did cerebral cortex PSDs. In the case of flunitrazepam, only one binding site was found for all four preparations, with cerebellar PSDs having twice the concentration of sites of cerebral PSDs. Photoaffinity labeling of the flunitrazepam receptor in PSDs resulted in the binding to a 51,000 Mr protein in both cases, with cerebellar PSDs again showing an increased concentration over that found in cerebral cortex PSDs. Based on this work, and on earlier work of ourselves and of others, we conclude that both populations of isolated PSDs contain inhibitory sites, but that the intact PSDs in both preparations are derived from Gray type I, probably excitatory, synapses, and that the inhibitory sites are found in the broken-up material in the PSD fractions which are derived from Gray type II, probably inhibitory, synapses.

Animals↗
PubMed1983

Plasticity in the central nervous system: do synapses divide?

Changes in the proportion of synapses containing postsynaptic densities with perforations during periods of increased synapse formation have led us to propose a hypothesis describing a possible division of preexisting synapses. Relevant features of this model are that various types of stimulation result in the following sequence of events: (i) the synaptic junction increases in area; (ii) a perforation forms in the enlarging synaptic junction; (iii) a synaptic spinule appears apposed to the perforation in the postsynaptic density; (iv) the perforation in the synaptic junction increases in size until the synaptic junction splits into two separate synaptic junctions within the same synaptic terminal; and (v) the dendritic spine divides into two, each containing a synaptic junction. Physiological responses in which synapse division may possibly play a role include hormone-induced neuronal changes, reinnervation of dendrites after lesions, and learning and memory.

Animals↗
PubMed1983

Identification of fodrin as a major calmodulin-binding protein in postsynaptic density preparations.

A major protein of postsynaptic densities (PSDs), a doublet of 230,000 and 235,000 Mr that becomes enriched in PSDs after treatment of synaptic membranes with 0.5% Triton X-100, has been found to be identical to fodrin (Levine, J., and M. Willard, 1981, J. Cell Biol. 90:631) by the following criteria. The upper bands of the PSD doublet and purified fodrin (alpha-fodrin) were found to be identical since both bands (a) co-migrated on SDS gels, (b) reacted with antifodrin, (c) bound calmodulin, and (d) had identical peptide maps after Staphylococcus aureus protease digestion. The lower bands of the PSD doublet and of purified fodrin (beta-fodrin) were found to be identical since both bands co-migrated on SDS gels and both had identical peptide maps after S. aureus protease digestion. The binding of calmodulin to alpha-fodrin was confirmed by cross-linking azido-125I-calmodulin to fodrin before running the protein on SDS gels. No binding of calmodulin to beta-fodrin was observed with either the gel overlay or azido-calmodulin techniques. A second calmodulin binding protein in the PSD has been found to be the proteolytic product of alpha-fodrin. This band (140,000 Mr), which can be created by treating fodrin with chymotrypsin, both binds calmodulin and reacts with antifodrin.

Actins↗
PubMed1982

Probes of eukaryotic DNA-dependent RNA polymerase II-II. Covalent binding of two purine nucleoside dialdehydes to the initiation subsite.

The catalytic center of wheat germ DNA-dependent RNA polymerase II (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) as a model eukaryotic enzyme system was probed with two purine nucleoside dialdehydes, 6-methylthioinosinedicarboxaldehyde (MMPR-OP) and a derivative 6-[(acetylaminoethyl)-1-naphthylamine-5-sulfonyl]thioinosinedicarboxaldehyde (AMPR-OP). Both drugs gave noncompetitive inhibition with respect to [3H]UMP incorporations into RNA, and inhibitor bindings were reversed with initiation substrates. The Ki values for MMPR-OP and AMPR-OP were determined to be 0.64 mM and 1.0 muM respectively. The drugs were covalently bound to the catalytic center by NaBH4 reduction. Both were found bound to the largest enzyme subunit, IIa. It is tentatively concluded that MMPR-OP and AMPR-OP inhibit RNA polymerase II by binding to an essential lysine in the initiation subsite of the catalytic center located on the IIa subunit.

DNA-Directed RNA Polymerases↗
PubMed1982

The cytoskeletal system of nucleated erythrocytes. II. presence of a high molecular weight calmodulin-binding protein.

Calmodulin was detected in dogfish erythrocyte lysates by means of phosphodiesterase activation. Anucleate dogfish erythrocyte cytoskeletons bound calmodulin. Binding of calmodulin was calcium-dependent, concentration-dependent, and saturable. Cytoskeletons consisted of a marginal band of microtubules containing primarily tubulin, and trans-marginal band material containing actin and spectrinlike proteins. Dogfish erythrocyte ghosts and cytoskeletons were found to contain a calcium-dependent calmodulin-binding protein, CBP, by two independent techniques: (a) 125I-calmodulin binding to cytoskeletal proteins separated by SDS PAGE, and (b) in situ azidocalmodulin binding in whole anucleate ghosts and cytoskeletons. CBP, with an apparent molecular weight of 245,000, co-migrated with the upper band of human and dogfish erythrocyte spectrin. CBP was present in anucleate ghosts devoid of marginal bands and absent from isolated marginal bands. CBP therefore appears to be localized in the trans-marginal band material and not in the marginal band. Similarities between CBP and high molecular weight calmodulin-binding proteins from mammalian species are discussed.

Animals↗
PubMed1982

Postmortem accumulation of tubulin in postsynaptic density preparations.

Postsynaptic density (PSD) preparations isolated from canine cerebral cortex that had been left at 0-37 degrees C for various times were found to become enriched in two bands in a time- but not temperature-dependent manner. The two bands were identified as tubulin subunits by gel mobility and immunology, Of all the isolated synaptic structures the increase in tubulin occurred primarily in the PSD fraction. The increase of tubulin also occurred in PSD preparations isolated from canine cerebellum and rat forebrain. Results obtained when PSD fractions were isolated from canine brain obtained as rapidly as possible after the death of the animal indicate that the maximum amount of tubulin in the PSD preparations is 2.5% of total Coomassie blue-stained protein as determined by scanning of gel electrophoretograms. These results imply that tubulin is probably not a major structural protein of the PSD as it exists in situ.

Animals↗
PubMed1981

Function of calmodulin in postsynaptic densities. I. Presence of a calmodulin-activatable cyclic nucleotide phosphodiesterase activity.

The postsynaptic density (PSD) fraction from canine cerebra cortex was found to contain an endogenous cyclic nucleotide-phosphodiesterase activity that was independent on Mn2+ and/or Mg2+ but not on Ca2+. Maximal activity was obtained at 1 micrometer Mn2+. This cyclic nucleotide phosphodiesterase activity was not decreased upon removal of the calmodulin from the PSD fraction, nor was it increased by the addition of calmodulin to a postsynaptic density fraction deficient in calmodulin. The enzymatic activity could be extracted by sonication, with the soluble enzyme having properties similar to those found in the native structure. Two peaks of cyclic nucleotide phosphodiesterase activities could be obtained after S-300 Sephacryl column chromatography of this soluble fraction: fraction I (excluded peak) and fraction II (215,000 mol wt). The fraction I activity preferred cyclic AMP over cyclic GMP and was not activated by calmodulin. The fraction II activity has an approximately fourfold lower Km for cyclic GMP over cyclic AMP. This fraction II activity was activatable by calmodulin, which increased the Vmax and decreased the Km in the case of both cyclic nucleotides. We conclude that two activities are present in the PSD, one activatable, and one not activatable, by calmodulin.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1981

Function of a calmodulin in postsynaptic densities. II. Presence of a calmodulin-activatable protein kinase activity.

Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity. As seen by autoradiographic analysis of coomassie blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [gamma-(32)P]ATP and Mg(2+) alone. Addition of Ca(2+) alone to the native PSD preparation had little or no effect on phosphorylation. However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 M(r). Similar results were also obtained in sonicated or freeze thawed native PSD preparations by addition of Ca(2+) alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions. The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulin-activatable phosphorylation, but not of the Mg(2+)-dependent activity, by the Ca(2+) chelator, EGTA, which also removes the calmodulin from the structure (26), and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca(2+). In addition, when a calmodulin-deficient PSD preparation was prepared (26), sonicated, and incubated with [gamma-(32)P]ATP, Mg(2+) and Ca(2+), one could not induce a Ca(2+)-stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins. Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 M(r) PSD protein is the one that is phosphorylated and not the 51,000 M(r) component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 M(r) protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg(2+)-dependent kinase(s). Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins I(a) and I(b), the cyclic AMP-dependent protein kinase, which definitely does phosphorylate protein I(a) and I(b) and is present in the PSD, does not phosphorylate the 51,000 and 62,000 M(r) proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter two proteins.

Animals↗
PubMed1981

Function of a calmodulin in postsynaptic densities. III. Calmodulin-binding proteins of the postsynaptic density.

A method has been developed for binding calmodulin, radioiodinated by the lactoperoxidase method, to denaturing gels and has been used to attempt to identify the calmodulin-binding proteins of cerebral cortex postsynaptic densities (PSDs). Calmodulin primarily bound to the major 51,000 Mr protein in a saturatable manner; secondarily bound to the 60,000 Mr region, 140,000 Mr region, and 230,000 Mr protein; and bound in lesser amounts to a number of other proteins. The major 51,000 Mr calmodulin-binding protein is one of unknown identity. Binding of iodinated calmodulin to these proteins was blocked by EDTA, EGTA, chlorpromazine, and preincubation with unlabeled calmodulin. Calmodulin iodinated by the chloramine-T method, which inactivates calmodulin did not bind to the PSD but bound nonspecifically to histone. Calmodulin did not bind to proteins from a variety of sources for which calmodulin interactions have not been found. Except for three proteins, all of the proteins of synaptic membranes that bind calmodulin could be accounted for by proteins of the PSD which are a part of the synaptic membrane fraction. The major 51,000 M, protein and the corresponding iodinated calmodulin binding were greatly reduced in cerebellar PSDs and this difference between cerebral cortex and cerebellar PSDs is discussed in light of the possible function of calmodulin in synaptic excitatory responses.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1980

Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities.

Postsynaptic densities (PSDs) have been isolated from cerebral cortex, midbrain, cerebellum, and brain stem by the Triton X-100 method previously used in the isolation of cerebral PSDs (Cohen et al., 1977, J. Cell Biol. 74:181). These PSDs have been compared in protein composition, protein phosphorylation, and morphology. Thin-section electron microscopy revealed that cerebral cortex and midbrain PSDs were identical, being approximately 57 nm thick and composed of apparent aggregates 20-30 nm in diameter. Isolated cerebellar PSDs appeared thinner (33 nm) than cerebral cortex PSDs and lacked the apparent 20- to 30-nm aggregates, but had a latticelike structure. In unidirectional and rotary-shadowed replicas, the cerebrum and midbrain PSDs were circular in shape with a large central perforation or hole in the center of them. Cerebellum PSDs did not have a large perforation, but did have numerous smaller perforations in a lattice like structure. Filaments (6-9 nm) were observed connecting possible 20- to 30-nm aggregates in cerebrum PSDs and were also observed radiating from one side of the PSD. Both cerebral cortex and midbrain PSDs exhibited identical protein patterns on SDS gel electrophoresis. In comparison, cerebellar PSDs (a) lacked the major 51,000 Mr protein, (b) contained two times less calmodulin, and (c) contained a unique protein at 73,000 Mr. Calcium plus calmodulin stimulated the phosphorylation of the 51,000 and 62,000 Mr bands in both cerebral cortex and midbrain PSDs. In cerebellar PSDs, only the 58,000 and 62,000 Mr bands were phosphorylated. In the PSDs from all brain regions, cAMP stimulated the phosphorylation of Protein Ia (73,000 Mr), Protein Ib (68.000 Mr), and a 60,000 Mr protein, although cerebrum and midbrain PSDs contained very much higher levels of phosphorylated protein than did the cerebellum. On the basis of the morphological criteria, it is possible that PSDs isolated from cerebrum and midbrain were derived from the Gray type I, or asymmetric, synapses, whereas cerebellum PSDs were derived from the Gray type II, or symmetric, synapses. Since there is some evidence that the type I synapses are involved in excitatory mechanisms while the type II are involved in inhibitory mechanisms, the role of the PSD and of some of its proteins in these synaptic responses is discussed.

Animals↗